The rise in antibiotic-resistant Mycobacterium tuberculosis and the lack of drugs capable of efficiently eradicating persistent bacilli responsible for life-long infections in humans emphasize the need for novel anti- TB agents with mechanisms of action different from those of existing drugs. The search for alternative drug therapies has led to our interest in class II fructose-1,6-bisphosphate aldolase (FBA), a key enzyme of glycolysis/gluconeogenesis that was shown to be induced in M. tuberculosis grown under oxygen-limiting conditions thought to mimic the physical microenvironment encountered by persistent bacilli in pulmonary lesions. Selective inhibition of FBA is expected to prevent M. tuberculosis from growing on host-derived fatty acids during persistent infection. Although ubiquitous in living organisms, FBAs can be divided into two classes which differ in their structure and reaction mechanism. While class I FBAs are the only type found in mammals, prokaryotes produce class II FBAs. The absence of class II FBAs from mammalian cells and the specificity of their structure and catalytic mechanism should make it possible to design specific inhibitors of class II enzymes that target pathogenic bacteria without affecting the host's gluconeogenetic/glycolytic pathways. We have designed for the first time two inhibitors that show activity against bacterial class II enzymes but low or no activity on class I enzymes in vitro. These fructose-1,6-bisphosphate analogs also showed bactericidal activity against actively replicating and non-replicating M. tuberculosis cells in vitro. The broad goal of this application is to validate M. tuberculosis class II FBA as a new target for drug discovery and its long-term goal is to design inhibitors of this enzyme. Using genetic approaches and our existing inhibitors, we will first investigate the essentiality of the fba gene in M. tuberculosis bacilli grown under different conditions. In the second part of our work, lipophilic versions of our inhibitors and other derivatives will be synthesized and tested against the purified FBA protein and M. tuberculosis whole cells. Information gained from this medicinal chemistry approach is expected to suggest extended strategies for FBA inhibition and guide the rational design of optimized inhibitors of this enzyme. PUBLIC HEALTH RELEVANCE: Novel drug targets whose inhibition would result in the killing of multi-drug-resistant as well as persistent Mycobacterium tuberculosis are urgently needed. We propose to validate the class II fructose 1,6 bisphosphate aldolase of the tubercle bacillus as such a therapeutic target and to synthesize inhibitors of this enzyme.